Cholesterol depletion reduces Helicobacter pylori CagA translocation and CagA-induced responses in AGS cells

Institute of Molecular and Cellular Biology & Department of Life Sciences, National Tsin Hua University, Hsinchu, Taiwan; Department of Microbiology, China Medical University, Taichung, Taiwan; Institute of Athletes, National Taiwan Sport University, Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan

^* To whom correspondence should be addressed. Email: wcwang{at}life.nthu.edu.tw.

	Abstract

Infection of Helicobacter pylori cagA-positive strains is associated with gastritis, ulcerations and gastric cancer. CagA is translocated into infected epithelial cells by a type IV secretion system and can be tyrosine phosphorylated, inducing signal transduction and motogenic responses in epithelial cells. Cellular cholesterol, a vital component of the membrane, contributes to membrane dynamics and functions and is important in VacA intoxication and phagocyte evasion during H. pylori infection. In this investigation, we have shown that cholesterol extraction by methyl--cyclodextrin (MCD) reduced the level of CagA translocation and phosphorylation. Confocal microscope visualization revealed that a significant portion of translocated CagA was colocalized with rafts marker GM1 and c-Src during infection. Moreover, GM1 was rapidly recruited into sites of bacterial attachment by live-cell imaging analysis. CagA and VacA were co-fractionated with detergent-resistant membranes (DRMs), suggesting that the distribution of CagA and VacA is associated with rafts in infected cells. Upon cholesterol depletion, their distribution shifted to non-DRMs. Accordingly, CagA-induced hummingbird phenotype and IL-8 induction were blocked by cholesterol depletion. Raft-disrupting agents did not influence bacterial adherence but did significantly reduce internalization activity in AGS cells. These results together suggest that delivery of CagA into epithelial cells by the bacterial type IV secretion system (TFSS) is mediated in a cholesterol-dependent manner.